Photo-crosslinked gellan gum-based hydrogels: methods and uses thereof

ABSTRACT

This invention refers to photo-crosslinked hydrogel materials based in gellan gum suitable for tissue engineering and regenerative medicine applications or as drug delivery systems. 
     Formulations of gellan gum with different degrees of acylation serve as precursor material for insertion of a polymerizable moiety. The materials are capable of free radical polymerization with a photo-initiator at mild temperatures and exposure to ultraviolet light, enabling control of reticulation and withstanding the encapsulation of human and animal cells and/or drugs, and any combination thereof. 
     The physicochemical and biological properties can be adjusted by combining different formulations of gellan gum and reaction conditions. 
     The matrix can be used either as an acellular or cellular system, dispensed manually or automatically by injection and crosslinked directly at the site of application, and can be processed using manual or automated systems in different types of scaffolds, such as hydrogels, fibres, 3D structures and micro- or nanoparticles.

TECHNICAL FIELD

The present invention relates to photo-crosslinked gellan gum hydrogelswhich are capable of undergoing free radical polymerization to be usedas biodegradable matrix in acellular and cellular systems for tissueengineering and regenerative medicine applications or as drug deliverysystems (DDS). Methods of modifying gellan gum to make it polymerizableand for hydrogel photo-crosslinking using certain sources of light areprovided. The system is biocompatible and could be dispensed manually orautomatically and polymerized in situ. These new hydrogels enable acontrolled reticulation process that withstands the encapsulation ofcells and/or drugs, creating versatile systems that can be applied inseveral settings of a tissue engineering approach.

STATE OF THE ART

Hydrogels present appealing properties for tissue engineering andregenerative medicine applications, and DDS: they swell and retain largeamounts of water, are tissue mimetic and can be delivered using littleinvasive procedures (i.e., injection). In addition, hydrogels provide ahighly hydrated microenvironment (resembling ECM) and they can beinjected and crosslinked in situ for allowing cell encapsulation andpreservation/induction of their differentiated phenotype. An injectablehydrogel should also present a sol-gel transition mechanism suitable forclinical purposes, i.e., it should be liquid to facilitate homogeneouscell distribution and injection, while being capable of rapidly gellingafter implantation. This unique combination of characteristics makesthem also useful in drug delivery applications.

Although a number of hydrogel systems based in natural polymers (e.g.,alginate and chitosan) have been developed to tackle, for example,cartilage regeneration, they still present several problems such asvariability of production, inability to be processed at mildtemperatures, poor water solubility, inadequate mechanical propertiesand uncontrolled degradation rates. We are proposing the use of a gellangum-based injectable system for the aforementioned applications. Thispolymer is an extracellular microbial polysaccharide that forms a gel inthe presence of metallic ions. Gellan gum has been studied forapplications in the cartilage tissue engineering field and furtherexperiments are ongoing to consolidate its therapeutic potential.

The chemical structure of gellan gum has been described as a linearanionic hetero-polysaccharide consisting of glucose-glucuronicacid-glucose-rhamnose as a repeating unit. This type of hydrogelpresents some interesting features that allow its use as an in vivoinjectable system. It is commercially available in two forms, acetylatedand deacetylated both forming thermo-reversible gels with differentmechanical properties in the presence of metallic ions and upontemperature decrease. It is acid and heat resistant and gel formationoccurs without the need of harsh reagents. Other advantageous featuresinclude the lack of cytotoxicity, some degree of bio-adhesiveness andthe existence of a free carboxylic group per repeating unit, which canbe used for improvement of functionalization. Moreover, gellan gumhydrogels have already been shown to adequately support the growth andECM deposition of human articular chondrocytes in vitro and in vivo.Gellan gum thermo-sensitive behaviour is suitable for injectableformulations since gelation can be performed in situ at a temperatureclose to body temperature. However, as verified for otherionic-crosslinked hydrogels, significant dissolution can occur in vivoand structural integrity may be lost over time. Work regarding gellangum modification, by addition of free radical polymerizable groups(namely acrylate and maleate groups), has been shown to enable theformation of a 3D biodegradable hydrogel network by polymerization undermild conditions.

Photo-polymerization has been used in recent work as an alternativemethod for hydrogel formation with increased structural and mechanicalintegrity. In this technique, polymers are modified with specificfunctional groups (e.g., methacrylates) that may undergo free radicalpolymerization in the presence of a photo-initiator at mild temperaturesand upon exposure to ultraviolet (UV) light. This polymerizationreaction induces the formation of covalent crosslinks between functionalmethacrylate groups along the backbone of the polymer chains. Afluid-solid phase transformation occurs then under physiologicalconditions, which is ideal for the rapid entrapment of cells in situ.Other advantages include significant temporal and spatial control of thegelation process and the possibility of using a clinically approved UVlight source and a biocompatible photo-initiator. Additionally, theinjected cell-loaded system is able to efficiently fill the irregulardefects at the site of injury, which is extremely important whenconsidering lesions at positions difficult to access.

The photo-polymerization of (meth)acrylates has been described fordental applications. In another work, it has been described the use of acalcium alginate hydrogel combined with methacrylated dextran to producea composite system suitable for drug delivery.

Several patents have been granted based in the use of photo-crosslinkedpolysaccharides hydrogels for different applications. The followingexamples should be taken into account by their relevance in the area ofthis invention:

U.S. Pat. No. 5,334,640 describes crosslinked biocompatible compositionsfor encapsulating biologic compounds. These compositions comprise atleast one ionically crosslinked component and one covalently crosslinkedcomponent. The crosslinkable mixtures and methods for encapsulation arealso provided.

U.S. Pat. No. 6,602,975 relates to photo-polymerizable biodegradablehydrogels for use as tissue adhesives and in controlled drug delivery.Hydrogels polymerized using free radical initiators under the influenceof long wavelength UV light, visible light excitation or thermal energyare described.

WO 2004029137 refers to functionalized chondroitin sulphate andcrosslinked polymer matrices comprising functionalized chondroitinsulphate to be used for tissue engineering, and specifically incartilage reconstruction. Inventors describe methods of obtaining andusing the functionalized polymer and the crosslinked matrices.

WO 2006036681 relates to compositions and methods for treating a tissuedefect. In particular, the invention describes a hydrogel that containsat least two functional groups, one which reacts with functional groupsfound in cartilage or bone, and the other which is reactive with thehydrogel. A method for applying the hydrogel to the cartilage surface isprovided.

US 20060057098 A1 describes a photo-reactive material which comprises apolysaccharide bound to a glycidyl ester via a covalent bond for theproduction of a photo-crosslinked polysaccharide by light exposure to beused in the medical field. The polysaccharide could be photo-crosslinkedleading to the formation of a 3D structure that retains water, i.e., ahydrogel.

U.S. Pat. No. 7,196,180 refers to methods for functionalization ofhyaluronic acid and crosslinking thereof to form hydrogels to be used astissue adhesive or separator, drug delivery system, matrix for cellcultures and temporary scaffold for tissue regeneration. The hyaluronicacid derivatives could be crosslinked in situ by reaction with differentfunctionalities or crosslinkers.

U.S. Pat. No. 7,365,059 describes a process for producing aphotocrosslinked-polysaccharide composition which is bounded to aphoto-reactive group and crosslinked by light-irradiation. The processdescribed comprises: freezing the photo-reactivepolysaccharide-containing solution, an aqueous solvent capable ofdissolving the photo-reactive polysaccharide, and any one substanceselected from the group consisting of alcohol having compatibility withthe aqueous solvent, a surfactant and a chelating agent; and irradiatingthe resulting frozen product with light.

WO 2009101518 A2 refers to the application of gellan gum forregenerative medicine and tissue engineering approaches, and focus onits processing methods and devices. Concerning this application, gellangum undergoes a controlled ionic crosslinking in the presence of aphosphate buffer, an acidic or alkaline solution.

SUMMARY

The present invention provides a photo-crosslinkable hydrogel systembased in gellan gum, thereby combining the most advantageouscharacteristics of the biomaterial with an increased temporal andspatial control of polymerization. This system, which also allows forlimited control of structural and mechanical properties of the material,is useful for tissue engineering and regenerative medicine applications,as well as for drug delivery.

The present invention describes a precursor for photo cross-linking ahydrogel (also called comprising photo cross-linking precursor) a gellangum which comprises:

-   -   at least one photo cross-linkable monomeric unit or subunit;    -   at least one monomeric unit or subunit chemically selective        functionality for binding, preferably at least one monomeric        unit or subunit chemically bounds to proteins, substances or        biomolecular analites and markers.

In a preferred embodiment, the gellan gum acylation degree is from noacyl groups up to two acyl substituents—acetate and glycerate—bothlocated on the same glucose residue, more preferably one glycerate perrepeat and one acetate per every two repeats.

A more preferred embodiment the precursor further comprising apolymerizable moiety such us a methacrylates, ethacrylates, itaconates,acrylamides, aldehydes or mixtures thereof.

In another preferred embodiment the precursor above described furthercomprises a photo-initiator selected from free-radical initiators suchas, methyl benzoylformate,2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone or mixturesthereof.

In a more preferred embodiment the precursor above described is photocross-linkable under aqueous conditions at any temperature between15-37° C., in particular at room or physiological temperature.

The present invention also discloses a hydrogel material comprisingprecursor for photo cross-linking a hydrogel based in gellan gun abovedescribed with water or aqueous solvent. In a more preferred embodimentthe precursor for photo cross-linking is between 0.01 to 2% (w/v), morepreferably 1 or 2% (w/v).

In a preferred embodiment the hydrogel further comprises aphoto-initiator selected from free-radical initiators.

The concentrations of photo-initiator concentrations could be between0.01 to 10 (w/v), preferably 0.1% or 0.05% (w/v).

A common photo-initiator could be methyl benzoylformate,2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone or mixturesthereof, among others.

In a preferred embodiment, the hydrogel is able to bephoto-polymerizable, namely by the use of ultraviolet rays and couldalso be present in the form of micro- or nanoparticles.

A more preferred embodiment of the present invention is a hydrogelmaterial that further comprises a second non photo cross-linked gellangum.

Another aspect of the present invention is a pharmaceutical compositioncomprising at least the precursor for photo cross-linking, a hydrogelobject of the present invention or the hydrogel material and furthercomprising a biological active agent, a therapeutic agent in amountspharmaceutically acceptable.

In a preferred embodiment the biological active agent is a cell, a stemcell, a protein, a therapeutic agent, a biomolecule, diagnostic markerand probe or a mixture thereof, encapsulated or not.

In another preferred embodiment the compositions could be present in theform of powder, aqueous solution or injectable solution.

Another aspect of the present invention is related with fibres,membranes, nets, gauzes or discs comprising the precursor for photocross-linking or the hydrogel, object of the present invention or thehydrogel material described in the present invention, with openinterconnected pores. These materials have a degree of permeabilityrange between 0.4-4000 (10⁻¹³ m⁴N⁻¹ s⁻¹), preferably between 0.4-400(10⁻¹³ m⁴N⁻¹ s⁻¹), more preferably between 0.4-100 (10⁻¹³ m⁴N⁻¹ s⁻¹).

Another embodiment of the present invention is a process for obtainingthe precursor for photo cross-linking the hydrogel, one of the objectsof the present invention, comprising the followings steps:

-   -   conversion of gellan gum with adequate degrees of acylation to a        photo-polymerizable material through the reaction with glycidyl        methacrylate. Preferably at controlled pH between 3-10,        preferably 8.5 and temperature between 18-90° C.;    -   crosslinking of the polymer by UV light in the presence of        different types of free radical initiators at mild temperatures.        Preferably the radical initiator is a photo-initiator.

The precursor for photo cross-linking a hydrogel, the hydrogel, thepharmaceuticals compositions and the materials described in the presentinventions could be used as biomaterial or/and in medicine.

Many different uses are possible as an injectable hidrogel solution; ascoating biosensor, prosthesis and implants, a conductor transmission gelor lubrificant in ultrasound equipments, in aesthetic medicine, inradio-frequency ablation medical procedures or as a nucleus pulposusimplant materials.

The materials are capable of free radical polymerization with aphoto-initiator at mild temperatures and exposure to ultraviolet light,enabling control of reticulation and withstanding the encapsulation ofhuman and animal cells and/or drugs, and any combination thereof.

The physicochemical and biological properties can be adjusted bycombining different formulations of gellan gum and reaction conditions.

The percursor matrix can be used either as an acellular or cellularsystem, dispensed manually or automatically by injection and crosslinkeddirectly at the site of application, and can be processed using manualor automated systems in different types of scaffolds, such as hydrogels,fibres, 3D structures and micro- or nanoparticles.

BRIEF DESCRIPTION OF THE FIGURES

The following figures provide preferred embodiments for illustrating thedescription and should not be seen as limiting the scope of invention.

FIG. 1 represents the strategy for the development of injectablephoto-crosslinkable scaffolds, which could be used with or withoutencapsulated cells for human tissue regeneration. The strategy is basedon methacrylate modification with glycidyl methacrylate (GMA) and insitu photo-polymerization of gellan gum.

FIG. 2 represents the schematic illustration of the pH-controlledreaction (pH 8.5) of glycidyl methacrylate with gellan gum (* indicatesthe repeating unit of low or high acyl gellan gum consisting ofglucose-glucuronic acid-glucose-rhamnose).

FIG. 3 illustrates the discs produced from methacrylated gellan gum (1day of reaction) after being a) equilibrated in a phosphate bufferedsaline (PBS) solution, or photo-crosslinked with: b) methylbenzoylformate (MBF) 0.1% (w/v) at 366 nm or c)2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (HHMPP) 0.050 (w/v)at 240-300 nm.

FIG. 4 represents the Fourier-transform infra-red (FTIR) of gellan gum,gellan gum-MA and photo-crosslinked gellan gum-MA. FIG. 4A shows thespectra for gellan gum and gellan gum methacrylated for 1 and 5 days ofreaction. FIG. 4B illustrates the spectra for methacrylated gellan gum(1 day reaction) and photo-crosslinked discs with MBF at 10 (w/v).

FIG. 5 represents the proton nuclear magnetic resonance (¹H-NMR) spectraof gellan gum (GG) and methacrylated gellan gum (GG-MA) performed in D₂Oat 70° C. The arrows indicate the peaks corresponding to the protonsnewly formed from the reaction of GG with GMA.

FIG. 6 represents the heating differential scanning calorimetry (DSC)curves of gellan gum, gellan gum-MA immersed in PBS andphoto-crosslinked gellan gum-MA discs. FIG. 6A illustrates the DSCcurves for gellan gum and methacrylated gellan gum discs equilibrated inPBS produced from methacrylated gellan gum after 1 and 5 days ofreaction. FIG. 6B shows the DSC curves for methacrylated gellan gumdiscs equilibrated in PBS and photo-crosslinked discs with MBF at 0.10(w/v).

FIG. 7 represents the dynamical mechanical analysis (DMA) of thedeveloped hydrogel discs (i.e., gellan gum, gellan gum-MA equilibratedin PBS and photo-crosslinked gellan gum-MA discs with MBF 0.10 (w/v) and0.050 (w/v) HHMPP). FIG. 7A shows the storage (E′) modulus and FIG. 7Brepresents the loss factor (tan δ) measured in PBS at 37° C.

FIG. 8 represents the weight loss (A) and water uptake (B) of gellan gumdiscs, gellan gum-MA discs equilibrated in PBS and photo-crosslinkeddiscs with MBF 0.10 (w/v) and 0.050 (w/v) HHMPP.

FIG. 9 graphically represents the cytotoxic evaluation of the leachablesreleased from the discs produced from gellan gum, methacrylated gellangum equilibrated in PBS and photo-crosslinked methacrylated gellan gum.

FIG. 10 illustrates cell viability of nucleus pulposus (NP) cellsisolated from rabbit intervertebral discs (IVDs) and encapsulated inphoto-crosslinked discs with 0.1% (w/v) MBF, after 3 and 14 days ofculturing (cells were stained with Calcein AM and observed underfluorescence microscopy).

DETAILED DESCRIPTION OF THE INVENTION

This invention provides photo-crosslinked gellan gum hydrogels, theirprocessing method and use in the field of tissue engineering andregenerative medicine or DDS. With several advantages for regenerativestrategies, the invented photo-crosslinkable gellan gum materialprovides the basis for the development of a new minimally-invasivesystem that could be used alone or seeded with cells suitable forrestoring, maintaining or enhancing tissue(s)/organ(s) function. Inaddition, the polymerizable system enables the conjugation withbioactive molecules and could be used for controlled delivery ofbiological molecules.

Gellan gum could present different degrees of acylation: the high acylform—presents two acyl substituents both located on the same glucoseresidue: on average, one glycerate per repeat and one acetate per everytwo repeats, produces soft, elastic, non-brittle gels; whereas the lowacyl form—presents no acyl groups and produces firm, non-elastic,brittle gels. The free carboxylic groups present in the structure ofgellan gum enable to confer improved functions. The insertion of apolymerizable moiety which may be selected, for example, frommethacrylates, ethacrylates, itaconates, acrylamides, and aldehydes,allows obtaining a photo-reactive hydrogel.

Gellan gum with different degrees of acylation and mixtures of bothisoforms were converted to a photo-polymerizable material through thereaction with glycidyl methacrylate at controlled pH (e.g., 3-10,preferably 8.5) and temperature (18-90° C.). Different degrees ofmethacrylation were obtained by varying the molar ratio of GMA to gellangum repeating unit and the reaction time (e.g., from 1 hour until 15days). The materials are biodegradable under physiological conditionsdue to hydrolysable bonds in the polymer backbone, resulting innon-toxic fragments that are easily removed from the body. The polymerwas crosslinked by UV light in the presence of different types ofphoto-initiators (e.g., type I, such as benzoin ethers, benzil ketals,alfa-Dialkoxy-aceto-phenones, alfa-Hydroxy-alkyl-phenones,alfa-Amino-alkyl-phenones, Acyl-phosphine oxides; and type II such asBenzo-phenones/amines, Thio-xantones/amines) selected from free-radicalinitiators and at mild temperatures, such as the photo-initiator methylbenzoylformate (MBF) and2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone (HHMPP). Theinsertion of methacrylate groups in gellan gum structure was verified byFourier-transform infra-red (FTIR) analysis and proton nuclear magneticresonance (¹H-NMR) spectroscopy performed before and afterpolymerization. The methacrylated gellan gum and photo-crosslinkedhydrogels were also characterized by differential scanning calorimetry(DSC) and dynamical mechanical analysis (DMA), and its degradation andswelling properties assessed in vitro. Moreover, in vitro cytotoxicityand proliferation/viability screening was performed.

The use of different formulations, preferentially 1:1, 1:2 and 2:1 ofGG-MA:photo-crosslinked GG, w:w % and reaction conditions (e.g., varyingthe initial GG concentration or molecular weight, varying the molarratio of GMA to gellan gum repeating unit, reaction time andtemperature, pH) allows for tailoring the final physicochemical andbiological properties of the hydrogels for specific applications. Oneaspect of the invention is to provide an injectable scaffold or DDS withtunable physicochemical and biological properties. Formulations of thephoto-crosslinkable hydrogels can use different gellan gum forms, i.e.,with different degrees of acylation. These will allow obtainingscaffolds possessing a wide range of physical properties (e.g.,strength, softness, flexibility, durability, degradability) according tothe desired use. Along with having adjustable properties, this systemalso provides the advantage of being dissolved in aqueous solutions atmild temperatures and allowing spatial and temporal control of hydrogelformation, which is dependent on the application of the crosslinkingagent.

These photo-crosslinked gellan gum-based hydrogels, used alone or incombination with bioactive species and/or cells, provide differentpossibilities for application in tissue engineering and regenerativemedicine strategies. Regarding their application in this area, thesematerials could be deposited by manual methods or using a 3D automaticdispensing device, being crosslinked before, during or after deposition.

Fibres of photo-crosslinked gellan gum-based are prepared with differentformulations of gellan gum-MA:MBF preferentially 1:0.01, 1:0.05 and1:0.1 w:w % in water, at room temperature. Then, an appropriate volumeof the mixture(s) are transferred to a device possessing a cylinder suchas a 20 mL syringe. By pressing the syringe piston, it is possible todosage the mixture through the movement along the longitudinal axis ofthe cylinder and through a needle of different shape and diameter, intoan aqueous medium. Finally, fibres possessing the diameter of the needleused are obtained by exposing the gel to UV light. Preferred, the devicemay be used to transfer the mixture into a water-free or hydrophobicsurface, a tri-dimensional mould with the shape of a fibre (e.g.,optical fibre), which comprise the UV light itself. This system allowsobtaining hollow fibres.

Preferred Synthesis of Methacrylated Gellan Gum

Preferably, the methacrylated gellan gum (gellan gum-MA) was prepared byreacting gellan gum with glycidyl methacrylate (FIG. 2). Gellan gum wasdissolved in distilled water at room temperature under constant stirringto obtain a final concentration of 10 (w/v). Different compositions ofcommercially available gellan gum were tested: 1) low acyl gellan gum(Gelzan®, Sigma); 2) high acyl gellan gum (Kelcogel® LT 100, CP Kelco)and 3) different mixtures of both isoforms. A homogeneous dispersion ofthe material was achieved after heating the solution to 90° C.Alternatively, prior to methacrylation reaction, gellan gum was dialyzedagainst deionized water for several days. After lyophilization, thepurified product was dissolved in distilled water to yield a 1% (w/v)solution. At room temperature, GMA (97%) was added to the solution atdifferent molar ratios to the repeating unit of gellan gum. The solutionwas adjusted to a pH of 8.5 with 1 M NaOH and the reaction occurred withvigorous stirring at room temperature for different periods of time. ThepH was automatically adjusted to 8.5 using 1 M NaOH during reaction.Different degrees of substitution were achieved either by varying themolar ratio of GMA and the reaction time. After each time point, thereaction products were precipitated with ½ volume of cold acetone andpurified by dialysis against deionized water for 4 days to removeresidual GMA. The purified products were frozen at −80° C. and recoveredby lyophilization.

Preferred Hydrogel Formation

Preferably, to produce photo-crosslinked gellan gum hydrogels, asolution of 2% (w/v) of gellan gum-MA was prepared in deionized water.The gellan gum-MA presents an advantage over non-methacrylated gellangum as it may be easily dissolved at body temperature (37° C.). Thephoto-initiator methyl benzoylformate (MBF) was added at differentconcentrations ranging from 0.05 to 10 (w/v). A different type ofphoto-initiator, 2-hydroxy-4′-(2-hydroxyethoxy)-2-methylpropiophenone(HHMPP) was also tested at concentrations between 0.05 and 10 (w/v).Gels with a cylindrical shape were prepared with a typical diameter of 6mm (FIG. 3). Methacrylated gellan gum hydrogel discs were also obtainedby immersion in a phosphate buffered saline (PBS) solution at pH 7.4.The photo-crosslinked gellan gum hydrogels were produced by exposing thegels to UV light for different time periods. The photo-crosslinked discscan be further equilibrated in PBS.

Characterization of Photo-Crosslinked Gellan Gum-Based HydrogelsFourier-Transform Infra-Red (FTIR) Analysis

The efficiency of methacrylation was evaluated before and afterphoto-crosslinking by FTIR analysis. The infrared spectra offreeze-dried samples were recorded at room temperature at a resolutionof 2 cm⁻¹ in the range 4400-800 cm⁻¹ for an average of 32 scans (FIG.4). FIG. 4A shows the FTIR spectra of gellan gum and gellan gummethacrylated for 1 and 5 days. In all spectra, typical gellan gumabsorption bands were found (i.e., 1618, 1412 and 1037 cm⁻¹). The FTIRanalysis revealed also the presence of new peaks at 1738 and 1645 cm⁻¹in the methacrylated polymer, which are characteristic of carbonylstretching vibration of an ester (νC=O) and double bound (νC=C)stretches, respectively. The appearance of new adsorption bands, whichare characteristic of GMA, indicates a successful incorporation ofmethacrylate groups in the structure of gellan gum.

FIG. 4B illustrates the FTIR spectra of photo-crosslinked discs producedin the presence of photo-initiator MBF at 1% (w/v). The FTIR analysisrevealed polymerization of methacrylate groups during UV exposuredemonstrated by the inexistence of the absorption peak at 1645 cm⁻¹ inthe photo-crosslinked discs. Moreover, it was observed a decrease in theabsorption band at 1738 cm⁻¹ identified in gellan gum methacrylated for1 day. The reduction of this band, assigned to carbonyl stretchingvibration of an ester (νC=O) from GMA, can be indicative of successfulpolymerization.

The results from FTIR analysis demonstrated that methacrylated gellangum was successfully produced and that this material is able to undergophoto-polymerization in the presence of the photo-initiator MBF.

¹H-NMR Spectroscopy

The insertion of methacrylate groups in gellan gum structure wascorroborated by ¹H-NMR analysis. FIG. 5 shows the ¹H-NMR spectra ofgellan gum and gellan gum-MA powders dissolved in deuterium-d₂ water(D₂O) recorded at 70° C. The D₂O peak at 4.3 ppm was used as reference.

The chemical shift for unmodified gellan gum presented characteristicsignals at 5.15 and 1.32 ppm, corresponding to H-1 and H-6 of theα-anomers of L-rhamnopyranosyl residue. Additionally, the signals at4.73 ppm and 4.55 ppm should be attributed to D-glucopyranosyl andD-glucuropyranosyl residues, respectively. These characteristic signalswere also present in the chemical shift for gellan gum-MA. The gellangum-MA spectra also showed the appearance of singlets at 1.96 (due tothe methyl proton of methacrylate), 5.77 and 6.18 ppm (both from thevinyl-proton), which are ascribed to the protons newly formed from thereaction of gellan gum with GMA.

These data clearly revealed that methacrylation was successfullyachieved.

DSC Analysis

Methacrylated gellan gum hydrogels (equilibrated in PBS) andphoto-crosslinked discs were produced at 2% (w/v) concentration fromgellan gum methacrylated for 1 and 5 days according to the proceduredescribed in preferred hydrogel formation. Gellan gum discs were alsoprepared at (w/v) concentration as described elsewhere by Oliveira etal. (J Biomed Mater Res A 2009), and were used as controls.

FIG. 6A displays the DSC profiles of gellan gum and gellan gummethacrylated for 1 and 5 days. The appearance of two or moreendothermic peaks in the heating DSC curves of methacrylated gellan gummay be an indication of the presence of junction zones with differentbonding energies or different rotational freedoms. Higher temperatureendothermic peaks, as observed for gellan gum-MA, could be attributed tothe melting of the zones with higher bonding energies or with lowerrotational freedoms.

FIG. 6B shows the DSC profile of photo-crosslinked discs produced in thepresence of photo-initiator MBF at 0.1% (w/v) using gellan gummethacrylated for 1 day. Once more, the appearance of multipleendothermic peaks in the heating DSC curves of photo-crosslinked gellangum suggests the existence of connections with different thermalstabilities.

The DSC results reveal that methacrylated gellan gum andphoto-crosslinked gellan gum-MA form ordered structures involving morethermally stable junction zones.

Dynamic Mechanical Analysis (DMA)

The mechanical behaviour of the modified gellan gum hydrogel discs wascharacterized by DMA. Methacrylated gellan gum hydrogels andphoto-crosslinked discs were produced at 2% (w/v) concentration fromgellan gum methacrylated for 1 day, according to preferred hydrogelformation. Gellan gum discs were also prepared at 2% (w/v) concentrationas described by Oliveira et al. (J Biomed Mater Res A 2009), and wereused as controls. Discs with 7 mm diameter and 4 mm height were producedby using a cylindrical silicone mould.

FIG. 7 shows the DMA of gellan gum-based hydrogel discs measured in wetstate (PBS) at 37° C., throughout a physiological relevant frequencyrange (0.1-15 Hz). The mechanical behaviour of gellan gum discs wascompared to methacrylated gellan gum and photo-crosslinked gellan gum-MAdiscs with either 0.10 (w/v) MBF or 0.050 (w/v) HHMPP. The storagemodulus of the hydrogels increased by means of increasing frequency, butit presented a viscoelastic-like behaviour (FIG. 7A). This means thatthe tested hydrogels are elastic in a certain extent. The storagemodulus at 1 Hz of photo-crosslinked gellan gum-MA discs with MBF(122.8±8.3 kPa) and HHMPP (151.2±29.9 kPa) is higher as compared to thatfor gellan gum hydrogels (56.2±1.4 kPa). This can be attributed to themore compact microstructure, i.e., higher crosslinking density. Thehigher crosslinking degree, the higher elasticity will present thehydrogels discs. Methacrylated gellan gum-MA hydrogels presented anintermediate behaviour (89.5±7.4 kPa).

Loss factor (tan δ) for gellan gum, methacrylated gellan gum andphoto-crosslinked gellan gum-MA discs is shown in FIG. 7B. Values variedbetween 0.15 and 0.21 at 1 Hz, with gellan gum displaying the highervalue. Differences in water retention and microstructure compaction ofthe hydrogels may explain these observations.

DMA results revealed that photo-crosslinked hydrogels display improvedmechanical properties. This data shows that mechanical performance canbe tailored by adjusting the reaction and processing conditions.

In Vitro Degradation and Swelling Capacity

Discs at 2% (w/v) made of gellan gum-MA equilibrated in PBS orphoto-crosslinked with 0.1% (w/v) MBF or 0.05% (w/v) HHMPP were preparedaccording to the procedure described in preferred hydrogel formation,and lyophilized afterwards. Gellan gum discs were also prepared at 2%(w/v) concentration as described by Oliveira et al. (J Biomed Mater ResA 2009), and were used as controls.

FIG. 8 shows the weight loss and water uptake ability of the developedhydrogels soaked in PBS (pH 7.4) at 37° C. under constant agitation (60rpm), for different time periods. Weight loss results showed that nosignificant degradation was observed for all the freeze-dried hydrogeldiscs tested (i.e., gellan gum, methacrylated gellan gum andphoto-crosslinked gellan gum-MA with MBF and HHMPP), after 30 days (FIG.8A). Despite, different weight loss profiles can be seen among thematerials.

Regarding the swelling ability of the hydrogels, the values for wateruptake in all the hydrogel discs obtained from gellan gum-MA (i.e.,methacrylated gellan gum-MA and photo-crosslinked gellan gum-MA with MBFand HHMPP) were lower as compared to those for gellan gum (FIG. 8B).This can be attributed to a higher crosslinking density, consistent witha tighter matrix, which thus is less able to swell.

These results indicate that methacrylated gellan gum enables to form amore structurally stable matrix, particularly by photo-polymerization.

Assessment of the Cytotoxicity of Photo-Crosslinked Gellan Gum-BasedHydrogels

The cytotoxicity of the photo-crosslinked hydrogels prepared frommethacrylated gellan gum after 1 day of reaction was assessed using animmortalized mouse lung fibroblasts cell line (L929) purchased fromEuropean Collection of Cell Cultures (ECACC, UK). L929 cells were grownas monolayer's in Dulbecco's modified Eagle's medium (DMEM) supplementedwith 100 (v/v) foetal bovine serum and (v/v) of anantibiotic-antimycotic mixture containing 10,000 U·mL⁻¹ penicillin Gsodium, 10,000 μg·mL⁻¹ streptomycin sulphate and 25 μg·mL⁻¹ amphotericinB as Fungizone® Antimycotic in 0.85% saline. The L929 cells wereincubated at 37° C. in a humidified atmosphere with 5% CO₂, and themedium changed every two days. The investigation of the potential effectof leachables released from the materials (within a 24 hours extractionperiod) on cellular metabolism was performed using a standard MTS(3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2(4-sulfofenyl)-2H-tetrazolium)viability test in accordance with ISO/EN 10993 part 5 guidelines. Thisassay is based on the bioreduction of the substrate MTS into a brownformazan product by dehydrogenase enzymes in metabolically active cells,being widely used for evaluation of cell viability. Discs at 2% (w/v)made of gellan gum-MA equilibrated in PBS or photo-crosslinked with 0.1%(w/v) MBF or 0.05% (w/v) HHMPP were prepared as previously describedunder sterile conditions in 48-well plates by adding a volume of 125 μLper well. Gellan gum discs with similar dimensions were also processedat 2% (w/v) as described by Oliveira et al. (J Biomed Mater Res A 2009),and used as controls. The materials were previously sterilized byethylene oxide. The extracts fluids were prepared according to theliterature. Briefly, a minimum of 41 discs with a diameter of 15 mm wereintroduced in 50 mL tubes containing 20 mL of complete DMEM culturemedium to produce the extracts. The tubes were incubated in athermostatic bath at 37° C. and 60 rpm for 24 hours. Confluent L929cells were detached from the culture flasks using trypsin (0.25%trypsin/EDTA solution) and a diluted cell suspension was prepared. Cellswere then seeded in each well of a 96-well plate (six replicates persample) at a density of 2×10⁴ cells per well. Afterwards, cells wereincubated for 24 hours at 37° C. in an atmosphere with 5% CO₂ forachieving 80-90% of confluence. The culture medium in each well wasremoved and replaced by an identical volume (200 μL) of the extractionfluids. After 1, 3 and 7 days, the extracts were removed and 300 μL of amixture containing serum-free culture medium without phenol red and MTS(CellTiter 96 One solution Cell Proliferation Assay kit) was added toeach well. After incubation for 3 hours at 37° C. and with 5% CO₂, theoptical density (OD) was measured at 490 nm using a plate reader. Alatex rubber extract was used as positive control for cell death andculture medium was used as a negative control representing the idealsituation for cell proliferation. The percentage of cell viability wascalculated after normalization with the mean OD value obtained for thenegative control. All tests were performed in triplicate. Statisticalanalysis was performed with GraphPad Prism using one-way analysis ofvariance followed by Bonferroni post-test and significance set at p<0.05for n=18.

The results obtained with MTS cytotoxicity test showed that L929 cellswere metabolically active after contacting with the different extractfluids for all the periods tested (FIG. 9). No statistically significantdifferences were found between the materials and negative control, andall the materials were considered to display a non-cytotoxic behavior.From the results, it can be concluded that the degree of methacrylationand the concentration of the photo-initiator (MBF or HHMPP) do not havea deleterious effect on cellular metabolism. It was also clearlyobserved a toxic effect of the positive control for cell death (latex)on cell viability (FIG. 9).

Biological Performance of Photo-Crosslinked Gellan Gum-Based Hydrogels

The ability of photo-crosslinked gellan gum-MA hydrogels to sustaincells viability was assessed using nucleus pulposus (NP) cells isolatedfrom New Zealand white rabbit intervertebral discs (IVDs). The NewZealand white rabbit is an outbred breed of the albino rabbit, middlesized animal often used for animal experiments, particularly for IVDregeneration studies. Rabbit NP cells were isolated using anenzymatic-based method (collagenase type II). Briefly, after animalsacrifice, the extracted discs were placed in PBS solution (pH 7.4) andwashed several times with PBS containing 1% (v/v) antibiotic-antimycoticmixture until total removal of blood or other bodily contaminants. NPtissue was then separated from any obvious dense annulus tissue andafter being washed several times with PBS containing 1% (v/v)antibiotic-antimycotic mixture, NP tissue digestion was performed byincubation at 37° C. in a humidified atmosphere of 5% CO₂ for 24 hoursin 10 mL of cell medium DMEM:F12 (1:1) supplemented with 10% (v/v) FBS,1% (v/v) of an antibiotic-antimycotic mixture and 0.01% (v/v)collagenase II. A cell strainer (100 μm) was used to separate the cellsfrom the remaining tissue debris. The isolated NP cells were expanded inDMEM:F12 (1:1) medium supplemented with 10% (v/v) FBS and 1% (v/v) of anantibiotic-antimycotic mixture until reaching confluence. After two cellpassages, cells were detached from the culture flasks using trypsin(0.25% trypsin/EDTA solution) and a diluted cell suspension was preparedand centrifuged at 1200 rpm for 5 minutes. For the encapsulation in thephoto-crosslinked hydrogel, the medium was completely aspirated and thecell pellet was re-suspended in gellan gum-MA hydrogel (methacrylatedfor 1 day) containing 0.1% (w/v) MBF. The mixture of cells and hydrogelwas centrifuged at 600 rpm for 3 minutes to allow homogenization ofcells. The mixture was then used to produce discs with a typicaldiameter of 7 mm using a silicone mould (200 μl per disc). The discswith encapsulated NP cells were then exposed to UV light to allowphoto-polymerization. Afterwards, the discs were incubated for differenttime periods in DMEM:F12 (1:1) medium.

Cell viability after specific times of culturing (3 and 14 days) wasassessed using Calcein AM staining. Briefly, a Calcein AM solution of1/1000 was prepared in culture medium. Photo-crosslinked discs withencapsulated rabbit NP cells were collected from the culturing platesand incubated in the Calcein AM solution for 15-30 min at 37° C. andafterwards washed in sterile PBS. The samples were then observed under afluorescence microscope.

Calcein AM staining showed a uniform distribution of viable NP cells inthe photo-crosslinked discs, after 3 and 14 days of culturing (FIG. 10).From images, it is possible to observe cells spreading which is anindication of cells activation. An increasing density of live cells wasobserved which demonstrate that these hydrogels supports cellsproliferation.

The invention is of course not in any way restricted to the embodimentsdescribed and a person with ordinary skill in the art will foresee manypossibilities to modifications thereof without departing from the basicidea of the invention as defined in the appended claims.

The following claims set out particular embodiments of the invention.

1. Precursor for photo cross-linking a hydrogel comprising a gellan gumwhich comprises: (a) at least one photo cross-linkable monomeric unit orsubunit; (b) at least one monomeric unit or subunit chemically selectivefunctionality for binding. 2-30. (canceled)